NAD(+)-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD(+)-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 x 10(-4) Weiss Unit mL(-1) to 0.4 Weiss Unit mL(-1) and the detection limit of 4.0 x 10(-4) Weiss Unit mL(-1).